The cellular slime mold Dictyostelium discoideum expresses three genes (sodA, sodB and sodC) encoding the extracellular Cu/Zn superoxide dismutases. Following H 2 O 2 treatment, the expression of sodA and sodB increased while that of sodC decreased. The sodC null strain formed multinucleate cells in a shaking culture. These results suggest that sodC plays a unique role in Dictyostelium discoideum.
Oxidative stress that results from the generation of reactive oxygen species is a significant source of cellular and DNA damage.1,2) The most serious oxidative damage is thought to be DNA lesions which can be mutagenic and thereby lead to genomic instability. Reactive oxygen species are produced endogenously as by-products of normal aerobic metabolism. The mitochondrial electron transport chain is the most prolific source of cellular reactive oxygen species.3) In addition, reactive oxygen species are produced by exposure to various environmental oxidants or UV-light. Some cellular toxicity from UV-light is mediated by the formation of reactive oxygen species near the cell membrane. 4)Organisms contain various enzymatic systems for detoxifying reactive oxygen species. One main defense system is provided by cytosolic Copper/Zinc superoxide dismutases (Cu/Zn SODs) that convert superoxide anions to H 2 O 2 , which is then converted to water by catalases or peroxidases. Saccharomyces cerevisiae mutants lacking cytosolic Cu/Zn SOD, encoded by SOD1, display certain aerobic growth defects 5) and hypersensitivity to oxidative stress. 6) In humans, cytosolic Cu/Zn SOD (SOD1) is a ubiquitous small cytosolic metalloenzyme providing protection against oxygen radicalinduced cellular damage. 7,8) Mutations in the SOD1 gene cause a familial form of amyotrophic lateral sclerosis (ALS), the most common adult motor neuron disease. 9,10) Cytosolic Cu/Zn SOD orthologs are widely distributed in eukaryotes, but have not been identified in the cellular slime mold Dictyostelium discoideum, which is a model organism in the study of cell biology and developmental biology.11) We show here the identification of the D. discoideum homolog of cytosolic Cu/Zn SOD, as well as the characterization of the gene disruption mutant. MATERIALS AND METHODSCell Lines and Culture Conditions D. discoideum Ax2 was used in its wild-type form. D. discoideum sodD null strain was established from Ax2, as described below. D. discoideum pslA null strain was established and stored in the laboratory of Dr. R. A. Firtel (University of California, San Diego). Ax2 was cultured in HL5 medium.12) The null strains were maintained in HL5 containing 5 mg/l blasticidin S (Funakoshi, Tokyo, Japan).Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis Expression kinetics of the genes were examined by RT-PCR using specific primers (sodD: 5Ј-CCAG-The apeA and apnA encode the Escherichia coli exonuclease III homolog and endonuclease IV homolog, respectively. 13,14) Expression of the actin15 coding for the Actin15 protein was examined as the experimental control. 15)The reactions were according to the manufacturer's instructions for the One Step RNA PCR Kit (Takara Shuzo, Kyoto, Japan). Reaction products were amplified 15 to 31 cycles after reverse transcription, and signal intensities were analyzed.Induction of Gene Expression Ax2 cells were plated on plain agar and incubated for 20 h. The multicellular structures formed on the plate were exposed to 254 nm UV-light (...
Genes for Copper/Zinc superoxide dismutases, designated sodA and sodB, in the cellular slime mold Dictyostelium discoideum were analyzed. It was found that these gene products contain charged amino acid residues and hydrophobic stretches in their N-terminal regions, suggesting that they are extracellular Cu/Zn superoxide dismutases. The sodA and sodB are expressed in cells in the growth phase and throughout the developmental phases, suggesting that they are the housekeeping genes. The sodA is induced by H(2)O(2) exposure but not by UV irradiation, whereas sodB is induced both by H(2)O(2) exposure and UV irradiation. Such distinct kinetics of the expression patterns suggests that these enzymes play unique roles in D. discoideum.
In the cellular slime mold Dictyostelium discoideum, Countin2 and Countin3 proteins are thought to limit the minimum size of the multicellular structure since countin2 -and countin3 -strains form small fruiting bodies. Expression levels of the cell-cell adhesion proteins, gp24 and gp80, in wild type and null mutants were compared. During the process of aggregation, countin2 -cells expressed lower levels of gp24 and gp80 than those expressed by the wild type. The expression levels of gp24 and gp80 in countin3 -cells were almost the same as those in wild-type cells in the early stages of aggregation but were lower than wild-type levels in the late stages. These results indicate that Countin2 and Countin3 proteins enhance the expression of gp24 and gp80 in D. discoideum in different ways.
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